InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Chemistry » Organic Chemistry » "Glycosylation", book edited by Stefana Petrescu, ISBN 978-953-51-0771-2, Published: September 26, 2012 under CC BY 3.0 license. © The Author(s).

Chapter 7

Glycosylating Toxin of Clostridium perfringens

By Masahiro Nagahama, Masataka Oda and Keiko Kobayashi
DOI: 10.5772/48112

Article top


Domain organization of clostridial glucosylating toxins.
Figure 1. Domain organization of clostridial glucosylating toxins.
Model of entry and intracellular modification of LCTs
Figure 2. Model of entry and intracellular modification of LCTs
Model of glucosyltransferase activity of LCTs.
Figure 3. Model of glucosyltransferase activity of LCTs.
Protein substrates and cosubstrates of the LCTs.
Figure 4. Protein substrates and cosubstrates of the LCTs.
Morphological changes of Vero cells upon treatment with SLO plus TpeL1-525.
Figure 5. Morphological changes of Vero cells upon treatment with SLO plus TpeL1-525.

Glycosylating Toxin of Clostridium perfringens

Masahiro Nagahama, Masataka Oda1 and Keiko Kobayashi1

1. Introduction

Clostridium perfringens type C strains that produce various toxins cause hemorrhagic noxious ulceration or mucousal necrosis of the small intestine in humans, pigs, cattle and chickens (Sakurai et al. 1997, Sakurai and Nagahama 2006). In humans, the bacteria cause necrotic enteritis, which is termed “pig-bel” (Sakurai and Nagahama 2006). C. perfringens has been classified into five types, A to E, according to the toxinogenicity of major extracellular toxins designated alpha-, beta-, epsilon- and iota-toxins. The C. perfringens strains defined as type C show alpha- and beta-, but not epsilon- and iota-toxigenicities (Sakurai and Nagahama 2006). Type C strains produce alpha-toxin, beta-toxin, beta2-toxin, and perfringolysin O. Beta-toxin is known to be the primary pathogenic factor of necrotic enteritis in the type C strains (Tweten 2005, Sakurai and Nagahama 2006). Beta2-toxin is a toxin discovered in C. perfringens type C isolated from piglets with necrotic enteritis, and speculated to be important because its gene has been detected in most C. perfringens type C strains recovered from animals with clinical disease (Manteca et al. 2002, Waters et al. 2003).

Most of the toxins produced by C. perfringens type C are toxic to particular cells or cell lines. Beta-toxin possesses lethal, dermonecrotic, and pressor activities. Direct evidence concerning the biological activities of beta-toxin had been lacking due to the absence of a susceptible cell line for experiments in vitro. However, a cell line that is susceptible to the toxin was found (Nagahama et al. 2003). The toxin induced swelling and lysis on HL60 cells by binding to lipid rafts and forming a functional oligomer. Beta2-toxins is lethal for mice and cytotoxic for the cell line I407 cells inducing cell rounding and lysis without affecting the actin cytoskeleton (Gibert et al. 1997). On the other hand, delta-toxin is toxic to various rabbit immune cells, i.e. alveolar macrophages, peritoneal appendix cells, bone marrow cells, splenocytes and thymocytes (Jolivet-Reynaud et al. 1982). Theta-toxin is the prototypic member of the cholesterol-dependent cytolysin (CDC) family that includes listeriolysin O (LLO), streptolysin O (SLO), pneumolysin, and others (Tweten 2005). Theta-toxin exhibits cytotoxic effects on macrophages to escape from phagosomes in consort with alpha-toxin. Alpha-toxin is essential for growth and spread of infection in the host (Sakurai et al. 2004), and it helps C. perfringens avoid the host defense mechanism by altering the normal traffic of the host phagocytes (Ochi et al. 2002, Oda et al. 2006). The role played by alpha-toxin in pathogenesis is dictated by its ability to interact with membranes, whether from outside the cell or while inside the phagosomes (Naylor et al. 1998). Alpha-toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis (Sakurai et al. 2004). On the other hand, enterotoxin, which has molecular masses of 35 kDa as a monomer (Duffy et al. 1982) and 90–200 kDa as aggregate forms with eukaryotic proteins (Singh et al. 2001), is known to be highly cytotoxic to Vero and Caco-2 cells (Gao and McClane 2012). Among the C. perfringens type C toxins reported previously, only enterotoxin is toxic to Vero cells, and no toxin is known to have a molecular mass of around 200 kDa without eukaryotic proteins.

TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. TpeL (named TpeL: C. perfringens large cytotoxin) is a truncated homologue of Clostridium difficile TcdA and TcdB (Amimoto et al. 2007). TpeL was identified in the culture supernatant of C. perfringens strain CP4 and is thought to be associated with necrotic enteritis (Amimoto et al. 2007). Coursodon et al. (2012) reported that TpeL may potentiate the effect of other virulence attributes of necrotic enteritis caused by C. perfringens.

2. The family of clostridial glucosylating toxins

Cytosolic mono-O-glucosylation is an important molecular mechanism by which various bacterial protein toxins and effectors target eukaryotic cells. C. difficile TcdA and TcdB, Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA) are important pathogenic factors of the family of large clostridial toxins (LCTs). The pathogenicity of C. difficile is based upon the action of at least one of the two major exotoxins (TcdA and TcdB) (Voth and Ballard 2005). TcdA and TcdB are the main cause of antibiotic-associated diarrhea and pseudomembraneous colitis (Voth and Ballard 2005), as a consequence of treatment with antibiotics, which destroy the normal microflora of the gut and allow colonization and proliferation of C. difficile bacteria (Bartlett et al. 1977, Kelly and LaMont 2008). Although the precise pathogenic mechanisms of induction of diarrhea and colitis are not known, it is generally accepted that the toxin-induced glucosylation of Rho GTPases is central to the action of the C. difficile toxins (Kelly and LaMont 2008). TcsL is implicated in toxic shock syndrome after medical-induced abortion (Ho et al. 2009) and TcnA causes gas gangrene syndrome (Tsokos et al. 2008). All these toxins are 50 to 90% identical in their amino acid sequences. They are large proteins of 250 to 308 kDa. This family has now more than 30 members, including putative glycosyltransferases from C. perfringens, Escherichia coli, Citrobacter rodentium, Photobacterium profundum, Pseudomonas fluorescens and various species of Chlamydia and Chlamidophila.

2.1. Structure of LCTs

LCTs are single protein chains containing four functional domains and share 26 to 76 % sequence identity and are structurally and functionally organized (Busch and Aktories 2000). LCTs are composed of four domains, the glucosylating enzymatic A-domain, the autocatalytic processing C-domain, the translocating D-domain and the binding B-domain (Fig. 1). The one third C-terminal part exhibits multiple repeated sequences (31 short repeats and 7 long repeats in TcdA), which are involved in the recognition of a cell-surface receptor. A trisaccharide (Gal-α1-3Gal-β1-4GlcNac) has been found to be the motif recognized by TcdA. Related carbohydrates could be involved as TcdA receptor. The gp96, a member heat shock protein family, has been proposed to bind TcdA to the plasma membrane of enterocytes (Na et al. 2008). Because this B-domain exhibits sequence similarity to the carbohydrate binding region of the glucosyltransferase from Streptococcus mutans, it was suggested early on that this part of the toxin is involved in binding to a carbohydrate-containing receptor. The crystal structure of the C-terminal binding domain of


Figure 1.

Domain organization of clostridial glucosylating toxins.

Clostridial glucosylating toxins are composed of four domains, the glucosylating enzymatic domain (A-domain), the autocatalytic processing domain (C-domain), the translocating domain (D-domain) and the binding domain (B-domain). The A-domain has the glucosyltransferase activity. The B-domain containing polypeptide repeats is responsible for specific receptor-binding. The C-domain participates in the auto-catalytic cleavage of LCTs and is a cysteine-protease with the catalytic residues DHC. InsP6 is required for activation of the cysteine protease. The D-domain is likely involved in the delivery of the A-domain into the cytosol. This domain includes a hydrophobic region suggested to be important for insertion of the toxin into endosome membranes.

TcdA has been determined (Ho et al. 2005, Greco et al. 2006), showing a solenoid-like structure with 32 repeats consisting of 15–21 amino acid residues and seven repeats consisting of 30 residues. The repeats form β-hairpins, arranged in pairs with each adjacent pair of hairpins rotated by 120° to the next pair, resulting in a screw-like structure of a left-handed β-solenoid helix (Greco et al. 2006). Co-crystallization with a derivate of the trisaccharide α-Gal(1,3)β-Gal(1,4)βGlcNAc confirmed the carbohydrate binding capacity of the domain. In this complex there are two carbohydrate-binding regions. However, in the full-length C-terminal fragment there are seven of these potential binding domains that are highly conserved, giving it a high binding capacity. Although there is little information about the binding domain of TcdB, it is believed that TcdB uses different receptors to bind to target cell surfaces than TcdA (Jank et al. 2007a).

The cysteine protease C-domain is located between residues 543 and 769 in TcdA and between residues 543 and 767 in TcdB (Egerer et al. 2007, Giesemann et al. 2008). It was shown that cleavage of the toxin occurs auto-catalytically by a cysteine protease activity, which is harbored in the C-domain, covering residues 544–955, directly down-stream of the glucosyltransferase domain (Egerer et al. 2007, Giesemann et al. 2008). Cys-698 and His-653 have been shown to be part of the catalytic dyad, which in addition to Asp-587 might participate in the auto-cleavage reaction (Egerer et al. 2007). The crystal structure of C-domain (543–809) from TcdA was resolved in the presence of inositol hexakisphosphate (InsP6) (Pruitt et al. 2009). InsP6 binds to the C-Domain, causing a conformational change that activates the auto-catalytic activity (Egerer et al. 2009). This locates between the enzymatic A-domain and the delivery D-domain, playing a role in proteolytic cleavage of the toxin. The toxins undergo autoproteolysis allowing only the enzymatic A-domain to be released into the cytosol in the presence of InsP6. Once the target cell has taken up the LCT via receptor-mediated endocytosis at the B-domain, the toxin undergoes autoproteolysis in order to allow the A-domain to pass across the endosomal membrane into the cytosol (Kreimeyer et al. 2011).

D-domain, which is located between residues 955 and 1852, is a large hydrophobic region that makes up almost 50% of the total size of the toxin (Barth et al. 2001, Qa'Dan et al. 2000). However, the exact function of D-domain is unknown. It is characterized by a hydrophobic stretch which is most probably responsible for membrane penetration (transmembrane prediction) (von Eichel-Streiber et al. 1992). Therefore, this region is speculated to as the translocation domain. Deletion studies proved the importance of the hydrophobic region for toxin activity (Barroso et al. 1994). A small region in the primary sequence between residues 965 and 1128 is characterized by hydrophobic amino acids and is suggested to play a role in formation of transmembrane structure during pore formation and translocation of the toxin into the cytosol (Voth and Ballard 2005). Pore formation induced by the toxin has been shown in artificial lipid membranes (Barth et al. 2001, Giesemann et al. 2006). However, so far it is not clear how pore formation relates to the delivery of the toxin into the cytosol.

The biologically active domain, A-domain, harboring the glucosyltransferase activity, is translocated into the cytosol, comprises the first 543 aa (Pfeifer et al. 2003, Rupnik et al. 2005). Therefore, cleavage of the toxin is required. The 3D-structure of A-domain showed that it was closely related to other bacterial glucosyltransferases belonging to the glucosyltransferase A family (Reinert et al. 2005). The catalytic core consists of 234 aa and is formed by a mixed α/β-fold with mostly parallel β-strands as the central part. The more than 300 additional residues are mainly helices, of which the first four N-terminal helices are most probably involved in membrane association, therefore assuring close proximity of the enzyme with its substrates. The structure of the central core is similar to that of glucosyltransferase A family (Reinert et al. 2005, Ziegler et al. 2008). Characteristic for glucosyltransferase A family members is the DXD motif involved in complexation of manganese ions, UDP and glucose. Mutation of these essential aspartate residues leads to inactivation of the toxin (Giesemann et al. 2008). The first aspartic residue of the DXD motif binds to ribosyl and glucosyl moieties of UDP-glucose and the second aspartic residues binds to divalent cation (mainly manganese ions) which increases the hydrolase activity and/or the binding of UDP-glucose (Just et al. 2000). Other amino acids in TcdB having an essential role in the enzymatic activity have been identified such as Trp-102 which is involved in the binding of UDP-glucose, Asp-270, Arg-273, Tyr-284, Asn-384, Trp-520, as well as Ile-383 and Glu-385 being important for the specific recognition of UDP-glucose (Busch et al 2000a and 2000b, Jank et al. 2005, Jank et al. 2007a,). Differences in α-helices probably account for the substrate specificity of each toxins (Reinert et al. 2005, Ziegler et al. 2008). Chimeric molecules between TcdB and TcsL have been used to identify the sites of Rho-GTPase recognition. Amino acids 408 to 468 of TcdB ensure the specificity for Rho, Rac and Cdc42, whereas in TcsL, the recognition of Rac and Cdc42 is mediated by residues 364 to 408, and that of Ras proteins by residues 408 to 516 (Voth and Ballard 2005, Jank et al. 2007a). The four N-terminal helices which mediates the binding of TcsL to phosphatidylserine, are possibly involved in membrane interaction (Mesmin et al. 2004). Amino acids 22-27 of Rho and Ras GTPase which are part of the transition of the α1-helix to the swich 1 region, are the main domain recognized by the glucosylating toxins (Müller et al. 1999). The cosubstrate for the bacterial glucosyltransferases is UDP-glucose; only TcnA utilizes UDP-N-acetylglucosamine (UDP-GlcNAc) (Selzer et al. 1996). This difference in cosubstrate specificity is based on sterical hindrance by bulky amino acids (e.g. Ile383/Gln385 in TcdB) blocking the catalytic pocket for the larger UDP-GlcNAc. In TcnA, small serine and alanine residues at the corresponding positions allow UDP-GlcNAc to enter the catalytic cleft (Jank et al. 2005). Little is known so far about the molecular/structural determinants underlying the differences in substrate recognition by different glucosylating toxins.

2.2. Internalization of LCTs

LCTs enter eukaryotic target cells through receptor-mediated endocytosis according to the ‘short trip model’ of bacterial exotoxin uptake (Sandvig et al. 2004). The cytotoxicity of the toxins are blocked by endosomal and lysosomal acidification inhibitors (monensin, bafilomycin A1, ammonium chloride) and the inhibiting effects can be by-passed by an extracellular acidic pulse (Popoff et al. 1996, Qa'Dan et al. 2000, Barth et al. 2001, Popoff and Bouvet 2009). As shown in Fig. 2, on binding to host cell receptors (Karlsson 1995, Giesemann et al. 2008), the toxins are endocytosed (Voth and Ballard 2005). After endocytosis, the acidification of early endosomes by the vesicular H+-ATPase induces a conformational change characterized by an increase in hydrophobicity, leading to membrane insertion (Qa'Dan et al. 2000, Barth et al. 2001, Voth and Ballard 2005). It reported that at low pH, LCTs induce channel formation in cell membranes and artificial lipid bilyers (Qa'Dan et al. 2000 and 2001, Giesemann et al. 2006). Membrane cholesterol seems critical for TcdA pore formation (Giesemann et al. 2006). Then, the hydrophobic region enables to form a pore through which the catalytic domain can translocate into the cytosol. Pore formation under acidic conditions has been demonstrated for TcdA and B (Barth et al. 2001, Giesemann et al. 2006). The exact mode of translocation remains to determine. The translocation-ligand domain remains associated with endosomal membranes and only the catalytic-DXD domain penetrates into the cytosol (Pfeifer et al. 2003, Rupnik et al. 2005). The N-terminal catalytic domain (A-domain) is then delivered from the early endosomes into the cytosol by auto-proteolytic activity stimulated by InsP6 (Reineke et al. 2007, Egerer et al. 2007). This autoproteolytic activity is induced by InsP6 and/or dithiothreitol and is responsible for the separation of the catalytic domain from the holotoxin (Egerer et al. 2007, Reineke et al. 2007). A cysteine protease domain (C-domain containing putative catalytic residues, DHC) has been identified close to the cutting site in TcdB (amino acid 544-955), which is conserved in all LCTs (Egerer et al. 2007, Reineke et al. 2007, Egerer et al. 2009). Cys-700, His-655 and Asp-589 have been identified as the catalytic triad. It reported that a cysteine protease catalytic triad is involved in processing of the toxin and auto-cleavage is essential for toxin activity (Egerer et al. 2007). Following its translocation and release, the catalytic-DXD fragment (A-domain) acts on its cytosolic targets, the GTPases of the Rho/Rac family, leading to the observed blockade of signal transduction processes and, consequently, the disaggregation of the cytoskeleton and cell death (Just and Gerhard 2004, Belyi and Aktories 2010). TcdA and B target Rho GTPases (Rho, Ras and Cdc42), which are molecular switches involved in numerous signal processes, in particular, the regulation of the actin cytoskeleton. Once the toxins enter the cytosol, they catalyse the addition of UDP-Glc (UDP-glucose) to Thr-37 (monoglucosylation) in Rho GTPase leading to depolymerization of actin filaments, disruption of the cytoskeleton and eventually cell rounding and cell death (Jank et al. 2007b, Belyi and Aktories 2010) (Fig. 2).

2.3. Glucosylation of Rho GTPase by LCTs

In the cytosol, LCTs glucosylate small GTPases of the Rho and Ras superfamily (Popoff et al. 1996, Selzer et al. 1996, Belyi and Aktories 2010). Small GTP-binding proteins involve organization of the cytoskeleton and control cellular activity of numerous other cellular enzymes. Rho proteins are molecular switches involved in various signal processes, including actin cytoskeleton regulation, cell cycle progression, gene transcription, and control of the activity of many enzymes like protein and lipid kinases, phospholipases, and nicotanimide adenine dinucleotide-oxidase (Etienne-Manneville and Hall 2002, Burridge and Wennerberg, 2004). In respect to their role in host–pathogen interactions, Rho proteins essentially participate in epithelial barrier functions and cell–cell contact, in immune cell

LCTs bind with their B-domain to the receptor of target cells. After endocytosis, the toxin inserts into the endosome membrane most likely involving the hydrophobic part of the D-domain. The acidic pH of the endosome triggers the first conformational change and results in pore formation of the ligand-translocation domain. Cytosolic InsP6 interacts with the cysteine protease C-domain and induces a second conformational change, activating the protease function. This results in cleavage of the toxin and release of the glucosyltransferase A-domain into the cytosol. In the cytosol, Rho GTPases are glucosylated and thereby inactivated. Inactive Rho can not interact with a numerous variety of effectors and induce multiple signaling events.


Figure 2.

Model of entry and intracellular modification of LCTs

Migration, phagocytosis, cytokine production, wound repair, immune cell signaling, and superoxide anion production. Modification of small GTP-binding proteins by LCTs arises at a Thr-35/37, depending on the Rho GTPase isoforms (Fig. 3) (Belyi and Aktories 2010). Marked differences in substrate specificity have been recognized among the various LCTs. TcdA and B glucosylate Rho, Rac and Cdc42 at Thr-37, whereas TcsL glucosylates Ras at Thr-35, Rap, Ral and Rac at Thr-37 and TcsH glucosylates Rho, Rac, Cdc42 (Fig. 4). LCTs catalyze the glucosylation of 21 kDa small GTP-binding proteins from UDP-glucose, except TcnA which uses UDP-N-acetylglucosamine as cosubstrate. TcnA glucosylates Rho, Rac, Cdc42 (Fig. 4). LCTs cleave the cosubstrate and transfer the glucose moiety to the acceptor amino acid of the Rho proteins (Popoff et al. 1996, Popoff and Bouvet 2009, Belyi and Aktories 2010) (Fig. 3). The conserved Thr, which is glucosylated, is located in switch 1. Thr-35/37 is involved in the coordination of Mg2+ and subsequently to the binding of the two phosphates of GTP. The hydroxyl group of Thr-35/37 is exposed to the surface of molecule in its GDP-bound


Figure 3.

Model of glucosyltransferase activity of LCTs.


Figure 4.

Protein substrates and cosubstrates of the LCTs.

TcdA: Clostridium difficile toxin A, TcdB: Clostridium difficile toxin B, TcsL: Clostridium sordellii lethal toxin, TcsH: Clostridium sordellii hemorrhagic toxin, TcnA: Clostridium novyi alpha-toxin, TpeL: Clostridium perfringnes large cytotoxin. U-Glc: UDP-glucose, U-NAG: UDP-N-acetylglucosamine form, which is the only accessible substrate of LCTs. Glucosylation of Rho or Ras GTPases inhibits activation of the GTPases by GEFs and blocks interaction with their effectors (Sehr et al. 1998, Vetter et al. 2000) as well as the cycling of Rho GTPases between the membrane localization and cytosolic localization (Belyi and Aktories 2010). Glucosylated Rho proteins are located at the membrane. Most importantly, the toxin-induced glucosylation inhibits the active conformation of Rho/Ras GTPases (Vetter et al. 2000, Geyer et al. 2003). Glucosylation of Thr-35 completely prevents the recognition of the downstream effector, blocking the G-protein in the inactive form (Popoff and Bouvet 2009).

2.4. Cellular effects of LCTs.

The inactivation of Rho proteins by LCTs-induced glucosylation causes extensive morphological changes, with loss of actin stress fibers, reorganization of the cortical actin, disrupution of the intercellular junctions and thus increase in cell barrier permeability. The actin cytoskeleton of intoxicated cells is redistributed, causing shrinking and rounding up of most cell types, which is initially accompanied by the formation of neurite-like retraction fibers. Finally, the retraction fibers disappear and the cells detach from the dishes (Ottlinger and Lin 1988, Popoff and Bouvet 2009). Inactivation of Rac assumes to be critical player in disorganization of actin cytoskeleton (Halabi-Cabezon et al. 2008). Numerous other cellular responses to the inactivation of Rho and Ras proteins by LCTs have been described, which are all caused by inhibition of the various functions of the small GTPase. They include inhibition of secretion (Prepens et al. 1996), phospholipase D activity (Schmidt et al. 1996), apoptosis (Brito et al. 2002, Voth and Ballard 2005), chemoattractant receptor signaling (Servant et al. 2000), phagocytosis (Caron and Hall 1998) and alteration of endothelial barrier function (Hippenstiel et al. 1997). However, the role of LCTs on pathogenesis including diarrhea and pseudomembranous colitis are still unknown.

Tissue damage and inhibition of the barrier function of enterocytes might explain the fluid response in toxin-induced diarrhea. TcdA and B affect the morphology and function of tight junctions and associated proteins (ZO-1, ZO-2, occuludin, claudin) to decrease transepithelial resistance, whereas E-cadherin junctions show little alteration (Nusrat et al. 2001, Chen et al. 2002, Aktories and Barbieri 2005). F-actin restruction is accompanied by the dissociation of occluding, ZO-1 and ZO-2 from lateral tight junctions without affecting adherens junctions. These data indicate that Rho proteins play an important role in tight junction regulation. On the other hand, TscH, which mainly modifies Rac, alter the permeability of intestinal cell monolayers causing a redistribution of E-cadherin whereas tight junctions are not significantly affected (Boehm et al. 2006).

TcdA and B induce apoptosis as a consequence of Rho glucosylation and caspase activation (Nottrott et al. 2007, Gerhard et al. 2008) or possibly cell necrosis (Genth et al. 2008). TcdB and TscH cause apoptosis by targeting mitochondria (Petit et al. 2003, Matarrese et al. 2007). In addition, the inactivation of Rho blocks various cellular functions including exocytosis, endocytosis, activation of lymphocyte, phagocytosis in macrophages, control of NADPH oxidase, activation of phospholipase D, contraction of smooth muscle, activation of the pro-apoptotic RhoB, and transcriptional activation via JNK/or p38 (Just and Gerhard 2004, Gerhard et al. 2005, Huelsenbeck et al. 2007, Popoff and Bouvet 2009). TcdA and B induce a large nflammatory response in the gut. There is massive infiltration of neutrophils, and release of many cytokines (Jefferson et al. 1999). TcdA-caused p38 activation induces the production of IL-8 and IL-1beta, necrosis of monocytes, and inflammation of intestinal mucosa (Popoff and Bouvet 2009). TcsL glycocylates Ras and inhibits the MAP-kinase cascade and PLD regulation. On the other hand, the role of those inhibition on cytotoxicity is still unclear (Schmidt et al. 1998, El Hadj et al. 1999).

3. Characterization of TpeL

C. perfringens type C has been identified as a causative agent of necrotizing enterocolitis associated with diarrhea and dysentery in infant animals (Tweten 2005, Sakurai and Nagahama 2006). Type C strain produces various toxins responsible for the pathogenesis (Sakurai and Nagahama 2006). Amimoto et al. (2007) fractionated the culture filtrate of C. perfringens type C strain MC18, and they discovered an unknown toxin (TpeL) that was lethal to mice. The toxin was cytotoxic to Vero cells, and its molecular mass was estimated to be about 180 kDa by SDS-PAGE analysis (Amimoto et al. 2007). These characteristics completely differed from the previously reported toxins. TpeL was purified with HPLC and affinity column coupled with the mAb. Coursodon et al. (2012) reported that TpeL-producing strains of C. perfringens type A are highly virulent for broiler chicks. Paredes-Sabja et al. (2011) reported that TpeL is also expressed during sporulation and is a sporulation-regulated C. perfringens toxin.

3.1. Toxicity of TpeL

TpeL was lethal to mice and toxic to Vero cells (Amimoto et al. 2007). The lethal activity of the purified TpeL in mice was determined as 62 MLD/mg (one was 16 μg) and 91 LD50/mg (one LD50 was 11 μg) by intravenous injection (Amimoto et al. 2007). TpeL showed obvious cytotoxicity in Vero cells, and the specific activity was 6.2 x 105 CU/mg (one CU was 1.6 ng) (Amimoto et al. 2007). Morphological changes induced by TpeL in Vero cells. The cytopathic effect induced by a low dose of TpeL was characterized by the enlargement of cells and appearance of rounded cells. Vero cells treated with a high dose of TpeL initially manifested similar changes to those treated with the low dose, then formed aggregates, and eventually detached from the well surface (Amimoto et al. 2007).

Vero cells were incubated with SLO (100 ng/ml) alone (A) or a combination of TpeL1-525 (10 μg/ml) with SLO (100 ng/ml) (B) at 37 °C for 15 min. Pictures were taken after 120 min of resealing. (C) Vero cells were incubated with various amounts of TpeL1-525 with SLO (100 ng/ml) at 37 °C for 15 min. After 120 min of resealing, pictures of cells were taken and the percentage of rounded cells was determined. Value of three experiments were given a mean ± the standard deviation (SD).

To clarify the biological acitivty of TpeL, we prepared a recombinant glycosyltransferase domain, TpeL1-525 (covering amino acids 1 to 525) because native TpeL is labile and is difficult to purify from the culture supernatant of C. perfringens type C, and the recombinant full-length TpeL was poorly expressed in Escherichia coli (Nagahama et al. 2011). As TpeL1-525 did not possess binding domain, we used the streptolysin O (SLO) delivery systems (Nagahama et al. 2011). As shown in Fig. 5(B), in the presence of SLO, TpeL1-525 caused the cell rounding like the native toxin. TpeL1-525 at 1 – 10 μg/ml in the presence of SLO induced cell rounding in a dose-dependent manner (Fig. 5(C)). The cells finally detached from the wells. On the other hands, the cells eventually detached from the well. Furthermore, when TpeL1-525 at a concentration of 1 to 10 μg/ml was delivered to the cells by SLO, cell viability decreased in a dose-dependent manner. The cytotoxicity was inhibited by the anti-TpeL antibody, and heat-inactivated TpeL1-525 was not cytotoxic. On the other hand, TpeL1-525 by itself did not have the cytotoxic effects. The results indicated that the N-terminal region of TpeL plays a role in the cytotoxicity and the C-terminal region is responsible for the binding of cells. The morphological alteration of cultured cells induced by TpeL is similar to that caused by TcdB and TcsL.


Figure 5.

Morphological changes of Vero cells upon treatment with SLO plus TpeL1-525.

3.2. tpeL gene

The gene encoding TpeL revealed the presence an ORF of 4953 bases (Amimoto et al. 2007). The tpeL gene encoded 1651 amino acid residues and the molecular mass of TpeL calculated from the deduced amino acid sequence was 191 kDa. A signal peptide region was not found within the ORF. The deduced amino acid sequence shared homology with TcdA, TcdB, TcsL and TcnA, called LCTs. The homology scores were 39 % to TcdA, 38 % to TcdB, 39% to TcsL and 30% to TcnA. The amino acid sequence of TpeL is shorter than that of any of these toxins, and the homologous region was located at the N-terminal site of the LCTs. A DXD motif in N-terminal region of LCTs is essential for glycosyltransferase activity, and W-102 in TcsL is an essential amino acid residue for the enzyme activity (Busch et al. 2000a). TpeL conserved the DXD motif and W-102 of TcsL. However, the C-terminal carbohydrate-binding sites of LCTs (von Eichel-Streiber et al. 1992) were not conserved.

The tpeL gene was detected not only in type C strains isolated in recent years but also ATCC 3626, a type B strain preserved for many years. It therefore suggest that the tpeL gene has been conserved in C. perfringens DNA for a long time. Interestingly, beta-toxin-gene-positive strains completely coincided with tpeL-positive strains among the 18 strains examined (Amimoto et al. 2007). Complete chromosomal and plasmid sequences of C. perfringens type A strain 13 are available (Shimizu et al. 2002). Also, there is no tpeL gene sequence within the data. It has been pointed out that beta- and epsilon-toxin genes carried by plasmids are sometimes lost during the passage of the strains (Katayama et al. 1996, Gibert et al. 1997). So, when the strain loses the plasmids, it changes to type A. In genetic study, total DNAs were used in the cloning and detection of the tpeL gene (Amimoto et al. 2007). Sayeed et al. (2010) reported that tpeL gene is located approximately 3 kb downstream of the plasmid-borne cpb gene. Gurjar et al. (2010) also reported that tpeL gene is localized to the plasmids containing cpb gene of cpe-negative type C isolates.

3.3. Glucosylation of small G proteins by TpeL

Rac1 is the only substrate GTPase inactivated by all LCTs. When the cells were treated with TpeL in the presence of SLO, glycosylation of cellular Rac1 was confirmed by Western blotting with the glycosylation-sensitive anti-Rac1 (Mab102) (Genth et al. 2006, Nagahama et al. 2011). TpeL and TcsL (Voth and Ballard 2005) act on Rac1 and the Ras subfamily but not RhoA. Furthermore, the isomeric TcdB from the variant C. difficile serotype F strain 1470 (TcdBF) that glucosylates Rac1 but not RhoA, has a cytopathic effect (Jank et al. 2007b). Halabi-Cabezon et al. (2008) reported that the glucosylation of Rac1 rather than RhoA correlates with the cytopathic effect of TcdB. It has been reported that Rac1 plays a critical role in the organization of the actin cytoskeleton (Bosco et al. 2009). These results strongly suggest that glycosylation of Rac1 is critical for the cytopathic effect of TpeL.

TpeL uses UDP-Glc and UDP-GlcNAc as sugar donors (Nagahama et al. 2011). All other LCTs use a single UDP-hexose (Belyi and Aktories 2010). The crystal structure provides evidence that two amino acids in the vicinity of the catalytic cleft are responsible for the specificity (Jank et al. 2005). TcdA and B, which both use UDP-Glc, have isoleucine and glutamine in the equivalent positions (Ile-383 and Gln-385 in TcdB), whereas TcnL, which uses UDP-GlcNAc, has serine and alanine residues at the respective positions (Jank et al. 2005). It has been reported that the bulkier side chains of Ile-383 and Gln-385 in TcdB limit the space of the catalytic cleft for the binding of UDP-GlcNAc and the exchange of these side chains with smaller groups changes the cosubstrate specificity from UDP-Glc to UDP-GlcNAc (Jank et al. 2005). TpeL has the smaller side chain Ala-383 and the bulkier side chain Gln-385 at the respective positions (Amimoto et al. 2007). We speculate that Ala-383 and Gln-385 in TpeL may stabilize the binding of UDP-Glc and UDP-GlcNAc and favor the acceptance of UDP-Glc and UDP-GlcNAc as the cosubstrates (Nagahama et al. 2011).

The sequential glycosylation of Rac1 by TpeL followed by TcdB, and vice versa indicates that both toxins share the same acceptor amino acid in Rac1. The acceptor amino acid of TcdB-glycosylated Rac1 has been determined as Thr-35 (Belyi and Aktories 2010). TpeL inactivates Rac1 through the glycosylation of Thr-35 (Nagahama et al. 2011).

TpeL glycosylated Rac1, as well as the Ras subfamily consisting of Ha-Ras, RalA, and Rap1B, but not RhoA and Cdc42 (Fig. 4). Important differences in substrate specificity have been detected among the various LCTs. Whereas TcdA, TcdB, and TcnA modify most RhoA, Rac1, and Cdc42 isoforms, TcsL glucosylates Rac1 but not RhoA or Cdc42 (Voth and Ballard 2005). On the other hand, TcsL also modifies the Ras subfamily, including Ras, Rap, and Ral isoforms (Voth and Ballard 2005). Thus, TpeL modifies similar substrates to TcsL. It was reported that Arg-455, Asp-461, Lys-463, and Glu-472 and residues of helix α17 (e.g., Glu-449) of TcdB are essential for enzyme-RhoA recognition (Jank et al. 2007b). Changing the respective amino acid residues in TcsL to those of TcdB reduced glycosylation of Ras by TcsL (Jank et al. 2007a). Furthermore, the introduction of helix α17 of TcdB into TcsL caused a reduction in the glycosylation of Ras subfamily proteins but permitted the glycosylation of RhoA, indicating that helix α17 is involved in RhoA's recognition by TcdB (Jank et al. 2007b). Glu-449, Lys-463, and Glu-472 in TcdB correspond to Lys, Arg, and Gly residues in TcsL and TpeL. Arg-455 in TcdB corresponds to Lys in TcsL and Gly in TpeL (Amimoto et al. 2007). The difference in those amino acid residues may be involved in recognizing small GTPases by TpeL. Additional residues in LCTs are needed for the recognition of small GTPases.

4. Conclusion

Infection with TpeL-positive C. perfringens strains may yield disease with a more rapid course and higher case fatality rate in broiler chicks. Thus, TpeL may potentiate the effect of other virulence attributes of necrotic enteritis strains of C. perfringens. TpeL from C. perfringens has been identified as a glycosyltransferase using UDP-GlcNAc and UDP-Glc as cosubstrates. The substrates of TpeL are confined to Rac1 and Ras subfamily proteins. The modification of Thr-35 on Rac1 induces cytopathic effects.


1 - K. Aktories, J. T. Barbieri, 2005Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol 3397410
2 - K. Amimoto, T. Noro, E. Oishi, M. Shimizu, 2007A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C. Microbiology 15311981206
3 - L. A. Barroso, J. S. Moncrief, D. M. Lyerly, T. D. Wilkins, 1994Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity. Microb Pathog 16297303
4 - H. Barth, G. Pfeifer, F. Hofmann, E. Maier, R. Benz, K. Aktories, 2001Low pH-induced formation of ion channels by Clostridium difficile Toxin B in target cells. J Biol Chem 2761067010676
5 - J. G. Bartlett, A. B. Onderdonk, R. L. Cisneros, D. L. Kasper, 1977Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J Infect Dis 136701705
6 - Y. Belyi, K. Aktories, 2010Bacterial toxin and effector glycosyltransferases. Biochim Biophys Acta 1800134143
7 - G. A. J. Brito, B. A. Fujji, A. A. Carneiro-Filho, T. Lima, Obrig, R. L. Guerrant, 2002Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells. J Infect Dis 18614381447
8 - C. Boehm, M. Gibert, B. Geny, M. R. Popoff, P. Rodriguez, 2006Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins. Cell Microbiol 810701085
9 - E. E. Bosco, J. C. Mulloy, Y. Zheng, 2009Rac1 GTPase: a “Rac” of all trades. Cell Mol Life Sci 66370374
10 - K. Burridge, K. Wennerberg, 2004Rho and Rac take center stage. Cell 116167
11 - C. Busch, K. Aktories, 2000Microbial toxins and the glucosylation of Rho family GTPases. Curr Opin Struct Biol 10528535
12 - C. Busch, F. Hofmann, R. Gerhard, K. Aktories, 2000aInvolvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases. J Biol Chem 2751322813234
13 - C. Busch, K. Schömig, F. Hofmann, K. Aktories, 2000bCharacterization of the catalytic domain of Clostridium novyi alpha-toxin. Infect Immun 6863786383
14 - E. Caron, A. Hall, 1998Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 28217171721
15 - M. L. Chen, C. Pothoulakis, J. T. La Mont, 2002Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A. J Biol Chem 27742474254
16 - C. F. Coursodon, R. D. Glock, K. L. Moore, K. K. Cooper, J. G. Songer, 2012TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks. Anaerobe 18117121
17 - L. K. Duffy, J. L. Mc Donel, B. A. Mc Clane, A. Kurosky, 1982Clostridium perfringens type A enterotoxin: characterization of the amino-terminal region. Infect Immun 38386388
18 - M. Egerer, T. Giesemann, T. Jank, K. J. Satchell, K. Aktories, 2007Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J Biol Chem 2822531425321
19 - M. Egerer, T. Giesemann, C. Herrmann, K. Aktories, 2009Autocatalytic processing of Clostridium difficile toxin B. Binding of inositol hexakisphosphate. J Biol Chem 28433893395
20 - N. B. El Hadj, M. R. Popoff, J. C. Marvaud, B. Payrastre, P. Boquet, B. Geny, 1999G-protein-stimulated phospholipase D activity is inhibited by lethal toxin from Clostridium sordellii in HL-60 cells. J Biol Chem 2741402114031
21 - S. Etienne-Manneville, A. Hall, 2002Rho GTPases in cell biology. Nature 420629
22 - Z. Gao, B. A. Mc Clane, 2012Use of Clostridium perfringens Enterotoxin and the Enterotoxin Receptor-Binding Domain (C-CPE) for Cancer Treatment: Opportunities and Challenges. J Toxicol 2012: 981626.
23 - H. Genth, J. Huelsenbeck, B. Hartmann, F. Hofmann, I. Just, R. Gerhard, 2006Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B. FEBS Lett 58035653569
24 - H. Genth, S. C. Dreger, J. Huelsenbeck, I. Just, 2008Clostridium difficile toxins: more than mere inhibitors of Rho proteins. Int J Biochem Cell Biol 40592597
25 - R. Gerhard, H. Tatge, H. Genth, T. Thum, J. Borlak, G. Fritz, I. Just, 2005Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB. J Biol Chem 28014991505
26 - R. Gerhard, S. Nottrott, J. Schoentaube, H. Tatge, A. Olling, I. Just, 2008Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J Med Microbiol 57765770
27 - M. Geyer, C. Wilde, J. Selzer, K. Aktories, H. R. Kalbitzer, 2003Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for the effector loop conformations observed by NMR spectroscopy. Biochemistry 42119511195
28 - M. Gibert, C. Jolivet-Reynaud, M. R. Popoff, 1997Beta2-toxin, a novel toxin produced by Clostridium perfringens. Gene 2036573
29 - T. Giesemann, T. Jank, R. Gerhard, E. Maier, I. Just, R. Benz, K. Aktories, 2006Cholesterol-dependent pore formation of Clostridium difficile toxin A. J Biol Chem 2811080810815
30 - T. Giesemann, M. Egerer, T. Jank, K. Aktories, 2008Processing of Clostridium difficile toxins. J Med Microbiol 57690696
31 - A. Greco, J. G. Ho, S. J. Lin, M. M. Palcic, M. Rupnik, K. K. Ng, 2006Carbohydrate recognition by Clostridium difficile toxin A. Nat Struct Mol Biol 13460461
32 - A. Gurjar, J. Li, B. A. Mc Clane, 2010Characterization of toxin plasmids in Clostridium perfringens type C isolates. Infect Immun 7848604869
33 - I. Halabi-Cabezon, J. Huelsenbeck, M. May, M. Ladwein, K. Rottner, I. Just, H. Genth, 2008Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1. FEBS Lett 58237513756
34 - S. Hippenstiel, S. Tannert-Otto, N. Vollrath, M. Krüll, I. Just, K. Aktories, C. , von Eichel-Streiber and N. Suttorp. (1997Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 272: L38L43.
35 - C. S. Ho, J. Bhatnagar, A. L. Cohen, J. K. Hacker, S. B. Zane, S. Reagan, M. Fischer, W. J. Shieh, J. Guarner, S. Ahmad, S. R. Zaki, L. C. Mc Donald, 2009Undiagnosed cases of fatal Clostridium-associated toxic shock in Californian women of childbearing age. Am J Obstet Gynecol 201: 459.e17
36 - J. G. Ho, A. Greco, M. Rupnik, K. K. Ng, 2005Crystal structure of receptor-binding C-terminal repeats from Clostridium difficile toxin A. Proc Natl Acad Sci USA 1021837318378
37 - J. Huelsenbeck, S. C. Dreger, R. Gerhard, G. Fritz, I. Just, H. Genth, 2007Upregulation of the immediate early gene product RhoB by exoenzyme C3 from Clostridium limosum and toxin B from Clostridium difficile. Biochemistry 4649234931
38 - T. Jank, D. J. Reinert, T. Giesemann, G. E. Schulz, K. Aktories, 2005Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis. J Biol Chem 2803783337838
39 - T. Jank, T. Giesemann, K. Aktories, 2007aClostridium difficile glucosyltransferase Toxin B-essential amino acids for substrate binding. J Biol. Chem 2823522235231
40 - T. Jank, T. Giesemann, K. Aktories, 2007bRho-glucosylating Clostridium difficile Toxins A and B: new insights into structure and function. Glycobiology 17: 15R-22R.
41 - K. K. Jefferson, M. F. Smith, Jr , D. A. Bobak, 1999Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes. J Immunol 16351835191
42 - C. Jolivet-Reynaud, J. M. Cavaillon, J. E. Alouf, 1982Selective cytotoxicity of Clostridium perfringens delta toxin on rabbit leukocytes. Infect Immun 38860
43 - I. Just, F. Hofmann, K. Aktories, 2000Molecular mechanism of action of the large clostridial cytotoxins, in Bacterial Protein Toxins. K. Aktories and I. Just, Editors. Springer, Berlin, 307331
44 - I. Just, R. Gerhard, 2004Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 1522347
45 - K. A. Karlsson, 1995Microbial recognition of target-cell glycoconjugates. Curr Opin Struc Bio 5622635
46 - S. Katayama, B. Dupuy, G. Daube, B. China, S. T. Cole, 1996Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne. Mol Gen Genet 251720726
47 - C. P. Kelly, J. T. La Mont, 2008Clostridium difficile- more difficult than ever. N Engl J Med 35919321940
48 - I. Kreimeyer, F. Euler, A. Marckscheffel, H. Tatge, A. Pich, A. Olling, J. Schwarz, I. Just, R. Gerhard, 2011Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile Toxin A. Naunyn Schmiedebergs Arch Pharmacol 383253262
49 - C. Manteca, G. Daube, T. Jauniaux, A. Linden, V. Pirson, J. Detilleux, A. Ginter, P. Coppe, A. Kaeckenbeeck, J. G. Mainil, 2002A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia? Vet Microbiol 86191202
50 - P. Matarrese, L. Falzano, A. Fabbri, L. Gambardella, C. Frank, B. Geny, M. R. Popoff, W. Malorni, C. Fiorentini, 2007Clostridium difficile toxin B causes apoptosis in epithelial cells by thrilling mitochondria. Involvement of ATP-sensitive mitochondrial potassium channels. J Biol Chem 28290299041
51 - B. Mesmin, K. Robbe, B. Geny, F. Luton, G. Brandolin, M. R. Popoff, B. Antonny, 2004A phosphatidylserine-binding site in the cytosolic fragment of Clostridium sordellii lethal toxin facilitates glucosylation of membrane-bound Rac and is required for cytotoxicity. J Biol Chem 2794987649882
52 - S. Müller, C. , von Eichel-Streiber and M. Moos. (1999Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864. Eur J Biochem 26610731080
53 - X. H. Na, M. P. Kim, C. Moyer, Pothoulakis, J. T. La Mont, 2008gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A. Infect Immun 7628622871
54 - M. Nagahama, S. Hayashi, S. Morimitsu, J. Sakurai, 2003Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells. J Biol Chem 2783693436941
55 - M. Nagahama, A. Ohkubo, M. Oda, K. Kobayashi, K. Amimoto, K. Miyamoto, J. Sakurai, 2011Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins. Infect Immun 79905910
56 - C. E. Naylor, J. T. Eaton, A. Howells, N. Justin, D. S. Moss, R. W. Titball, A. K. Basak, 1998Structure of the key toxin in gas gangrene. Nat Struc. Biol 5738746
57 - S. Nottrott, J. Schoentaube, H. Genth, J. Just, R. Gerhard, 2007Clostridium difficile toxin A-induced apoptosis is 53but depends on glucosylation of Rho GTPases. Apoptosis 12: 1443-1453.
58 - A. Nusrat, C. von, C. J. R. Eichel-Streiber, P. Turner, J. L. Verkade, Madara, C. A. Parkos, 2001Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 6913291336
59 - S. Ochi, T. Miyawaki, H. Matsuda, M. Oda, M. Nagahama, J. Sakurai, 2002Clostridium perfringens alpha-toxin induces rabbit neutrophil adhesion. Microbiology 148237
60 - M. Oda, S. Ikari, T. Matsuno, Y. Morimune, M. Nagahama, J. Sakurai, 2006Signal transduction mechanism involved in Clostridium perfringens alpha-toxin-induced superoxide anion generation in rabbit neutrophils. Infect Immun 742876
61 - M. E. Ottlinger, S. Lin, 1988Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells. Exp Cell Res 174215229
62 - D. Paredes-Sabja, N. Sarker, M. R. Sarker, 2011Clostridium perfringens tpeL is expressed during sporulation. Microb Pathog 51384388
63 - P. Petit, J. Bréard, V. Montalescot, N. B. El Hadj, T. Levade, M. Popoff, B. Geny, 2003Lethal toxin from Clostridium sordellii induces apoptotic cell death by disruption of mitochondrial homeostasis in HL-60 cells. Cell Microbiol 5761771
64 - G. Pfeifer, J. Schirmer, J. Leemhuis, C. Busch, D. K. Meyer, K. Aktories, H. Barth, 2003Cellular uptake of Clostridium difficile toxin B: translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J Biol Chem 2784453544541
65 - M. R. Popoff, F. Chaves-Olarte, E. Femichez, C. von, M. Eichel-Streiber, P. Thelestam, D. Chardin, B. Cussac, P. Antonny, G. Chavrier, M. Flatau, J. Giry, de Gunzburg and P. Boquet. (1996Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J Biol Chem 2711021710224
66 - M. R. Popoff, P. Bouvet, 2009Clostridial toxins. Future Microbiol 410211064
67 - U. Prepens, I. Just, C. , von Eichel-Streiber and K. Aktories. (1996Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase). J Biol Chem 27173247329
68 - R. N. Pruitt, B. Chagot, M. Cover, W. J. Chazin, B. Spiller, D. B. Lacy, 2009Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile Toxin A. J Biol Chem 2842193421940
69 - M. Qa’Dan, L. M. Spyres, J. D. Ballard, 2000pH-induced conformational changes in Clostridium difficile Toxin B. Infect Immun 6824702474
70 - M. Qa’Dan, L. M. Spyres, J. D. Ballard, 2001pH-enhanced cytopathic effects of Clostridium sordellii lethal toxin. Infect Immun 6954875493
71 - J. Reineke, S. Tenzer, M. Rupnik, A. Koschinski, O. Hasselmayer, A. Schrattenholz, H. Schild, C. von, Eichel-Streiber, 2007Autocatalytic cleavage of Clostridium difficile toxin B. Nature 446415419
72 - D. J. Reinert, T. Jank, K. Aktories, G. E. Schulz, 2005Structural basis for the function of Clostridium difficile toxin B. J Mol Biol 351973981
73 - M. Rupnik, S. Pabst, M. Rupnik, C. von, H. Eichel-Streiber, Urlaub, H. D. Soling, 2005Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells. Microbiology 151199208
74 - J. Sakurai, M. Nagahama, S. Ochi, 1997Major toxins of Clostridium perfringens. J Toxicol Toxin Rev 16195214
75 - J. Sakurai, M. Nagahama, M. Oda, 2004Clostridium perfringens alpha-toxin: characterization and mode of action. J Biochem 136569574
76 - J. Sakurai, M. Nagahama, 2006Clostridium perfringens beta-toxin: characterization and action. Toxin Rev 2589108
77 - K. Sandvig, B. Spilsberg, S. U. Lauvrak, M. L. Torgersen, T. G. Iversen, B. van Deurs, 2004Pathways followed by protein toxins into cells. Int J Med Microbiol 293483
78 - S. Sayeed, J. Li, B. A. Mc Clane, 2010Characterization of virulence plasmid diversity among Clostridium perfringens type B isolates. Infect Immun 78495504
79 - M. Schmidt, U. Rümenapp, C. Bienek, J. Keller, C. , von Eichel-Streiber and K. H. Jakobs. (1996Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins. J Biol Chem 27124222426
80 - M. Schmidt, M. Voss, M. Thiel, B. Bauer, A. Grannass, E. Tapp, R. H. Cool, J. de Gunzburg, C. von, Eichel-Streiber, K. H. Jakobs, 1998Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. J Biol Chem 27374137422
81 - P. Sehr, G. Joseph, H. Genth, I. Just, E. Pick, K. Aktories, 1998Glucosylation and ADP-ribosylation of Rho proteins- Effects on nucleotide binding, GTPase activity, and effector-coupling. Biochemistry 3752965304
82 - J. Selzer, F. Hofmann, G. Rex, M. Wilm, M. Mann, I. Just, K. Aktories, 1996Clostridium novyi α-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins. J Biol Chem 2712517325177
83 - G. Servant, O. D. Weiner, P. Herzmark, T. Balla, J. W. Sedat, H. R. Bourne, 2000Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 28710371040
84 - T. Shimizu, K. Ohtani, H. Hirakawa, K. Ohshima, A. Yamashita, T. Shiba, N. Ogasawara, M. Hattori, S. Kuhara, H. Hayashi, 2002Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 999961001
85 - U. Singh, L. L. Mitic, E. U. Wieckowski, J. M. Anderson, B. A. Mc Clane, 2001Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells. J Biol Chem 2763340233412
86 - M. S. Tsokos, F. Schalinski, J. P. Paulsen, K. Sperhake, Puschel, I. Sobottka, 2008Pathology of fatal traumatic and nontraumatic clostridial gas gangrene: a histopathological, immunohistochemical, and ultrastructural study of six autopsy cases. Int J Legal Med 1223541
87 - R. K. Tweten, 2005Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infect Immun 7361996209
88 - I. R. Vetter, F. Hofmann, S. Wohlgemuth, C. Herrmann, I. Just, 2000Structural consequences of mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin. J Mol Biol 30110911095
89 - Eichel. von-Streiber, C. , M. Sauerborn, H. K. Kuramitsu, 1992Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases. J Bacteriol 17467076710
90 - D. E. Voth, J. D. Ballard, 2005Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18247263
91 - M. Waters, A. Savoie, H. S. Garmory, D. Bueschel, M. R. Popoff, J. G. Songer, R. W. Titball, B. A. Mc Clane, M. R. Sarker, 2003Genotyping and phenotyping of beta2-toxigenic Clostridium perfringens fecal isolates associated with gastrointestinal diseases in piglets. J Clin Microbiol 4135843591
92 - M. O. Ziegler, T. Jank, K. Aktories, G. E. Schulz, 2008Conformational changes and reaction of clostridial glycosylating toxins. J Mol Biol 37713461356